Solid phase synthesis of LXR ligands

ABSTRACT

The invention provides solid support synthetic methods for producing combinatorial libraries of modulators of LXRs. The combinatorial libraries thus produced are useful both as diagnostic indicators of LXRα function and as pharmacologically active agents. The combinatorial libraries find particular use in the treatment of disease states associated with cholesterol metabolism, particularly atherosclerosis and hypercholesterolemia.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/194,911, filed Apr. 5, 2000, which is hereby incorporated byreference in its entirety for all purposes.

FIELD OF THE INVENTION

[0002] The present invention relates to solid phase synthetic methods,and more particularly, to solid support synthetic methods useful forproducing combinatorial libraries of modulators of LXRs.

BACKGROUND OF THE INVENTION

[0003] Cholesterol is used for the synthesis of bile acids in the liver,the manufacture and repair of cell membranes, and the synthesis ofsteroid hormones. There are both exogenous and endogenous sources ofcholesterol. The average American consumes about 450 mg of cholesteroleach day and produces an additional 500 to 1,000 mg in the liver andother tissues. Another source is the 500 to 1,000 mg of biliarycholesterol that is secreted into the intestine daily; about 50 percentis reabsorbed (enterohepatic circulation). Excess accumulation ofcholesterol in the arterial walls can result in atherosclerosis that ischaracterized by plaque formation. The plaques inhibit blood flow andpromote clot formation, and can ultimately cause heart attacks, strokeand claudication. Development of therapeutic agents for the treatment ofatherosclerosis and other diseases associated with cholesterolmetabolism has been focused on achieving a more complete understandingof the biochemical pathways involved. Most recently, liver X receptors(LXRs) were identified as key components in cholesterol homeostasis.

[0004] The LXRs were first identified as orphan members of the nuclearreceptor superfamily whose ligands and functions were unknown. Two LXRproteins α and β are known to exist in mammals.. The expression of LXRαis restricted, with the highest levels being found in the liver, andlower levels found in kidney, intestine, spleen, and adrenals. See,Willy, et al., Genes Dev. 9(9):1033-45 (1995). LXRβ is ratherubiquitous, being found in nearly all tissues examined. Recent studieson the LXRs indicate that they are activated by certain naturallyoccurring, oxidized derivatives of cholesterol, including22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and24,25(S)-epoxycholesterol. See, Lehmann, et al., J. Biol. Chem.272(6):3137-3140 (1997). The expression pattern of LXRs and theiroxysterol ligands provided the first hint that these receptors may playa role in cholesterol metabolism. See, Janowski, et al., Nature383:728-731 (1996).

[0005] As noted above, cholesterol metabolism in mammals occurs viaconversion into steroid hormones or bile acids. The role of LXRs incholesterol homeostasis was first postulated to involve the pathway ofbile acid synthesis, in which cholesterol 7α-hydroxylase (CYP7α)operates in a rate-limiting manner. Support for this proposal wasprovided when additional experiments found that the CYP7α promotercontained a functional LXR response element that could be activated byRXR/LXR heterodimers in an oxysterol- and retinoid-dependent manner.

[0006] Confirmation of LXR function as a transcriptional control pointin cholesterol metabolism was made using knockout mice, particularlythose lacking LXRαSee, Peet, et al., Cell 93:693-704 (1998). Micelacking the receptor LXRα (e.g., knockout or (−/−) mice) lost theirability to respond normally to increases in dietary cholesterol and wereunable to tolerate any cholesterol in excess of that synthesized denovo. LXRα (−/−) mice did not induce transcription of the gene encodingCYP7α when fed diets containing additional cholesterol. This resulted inan accumulation of large amounts of cholesterol in the livers of LXRα(−/−) mice, and impaired hepatic function. These results furtherestablished the role of LXRα as the essential regulatory component ofcholesterol homeostasis. LXRα is also believed to be involved in fattyacid synthesis. Accordingly, the discovery of new LXRα modulators suchas antagonists, via screening methods could provide treatment for avariety of lipid disorders including obesity and diabetes.

[0007] High-throughput screening techniques allow for assaying theactivity of thousands of molecules in short order. However, if moleculescan only be synthesized one at a time, the rate of molecule submissionto the assay becomes the rate-limiting step. To remedy this situation,various combinatorial techniques have been devised and implemented.Combinatorial chemistry is defined as the repetitive and systematiccovalent attachment of different structural moieties to one another toproduce a mixture of numerous distinct molecular entities or targetmolecules (i.e., combinatorial libraries). The desired target moleculesinclude peptides, oligonucleotides, and small organic molecules. Ingeneral, combinatorial chemistry is utilized to generate a group ofstructurally related analogs that can then be evaluated to establishstructure-activity relationships (SAR) and to optimize biologicalpotency.

[0008] The importance of LXRs, and particularly LXRα, to the delicatebalance of cholesterol metabolism and fatty acid biosynthesis has led tothe development of modulators of LXRs which are useful as therapeuticagents or diagnostic agents for the treatment of disorders associatedwith bile acid and cholesterol metabolism, including cholesterolgallstones, atherosclerosis, lipid storage diseases, obesity, anddiabetes (see, co-pending application Ser. No. 09/479315, filed Jan. 6,2000, incorporated herein by reference for all purposes). In view of theforegoing, there is a need in the art for combinatorial libraries of LXRmodulators and the methods to produce them. The present inventionfulfills this and other needs.

SUMMARY OF THE INVENTION

[0009] The importance of LXRs, and particularly LXRα to the delicatebalance of cholesterol metabolism and fatty acid biosynthesis has led tothe development of modulators of LXRs which are useful as therapeuticagents or diagnostic agents for the treatment of disorders associatedwith bile acid and cholesterol metabolism. However, more efficaciouscompounds are needed. As such, the present invention provides a methodfor preparing LXR ligands on a solid support, comprising:

[0010] (a) attaching an aniline derivative to the solid support toprovide a support-bound aniline derivative;

[0011] (b) contacting the support-bound aniline derivative with analdehyde or ketone under reductively aminating conditions to provide asupport-bound substituted aniline derivative; and

[0012] (c) contacting the support-bound substituted aniline derivativewith an acylating agent to provide an LXR ligand on the solid support.In certain preferred embodiments, the LXR ligand or modulator is cleavedor removed from the solid support.

[0013] The methods of the present invention enable the efficientgeneration of modulators of LXRs and other amide-derived productsfollowing cleavage from the support. In addition, the methods of thepresent invention can be used to generate diverse N-substitutedbenzanilide derivatives which, in turn, may be used in the formation ofcombinatorial libraries of compounds that can subsequently be screenedfor biological activity.

[0014] As such, the present invention provides a combinatorial librarycomprising compounds having the formula:

[0015] wherein R¹ is a group including, but not limited to, optionallysubstituted alkyl, optionally substituted aryl, optionally substituted(C₈-C₁₈)bicycloalkyl, optionally substituted (C₈-C₁₈)tricycloalkyl,optionally substituted (C₈-C₁₈)heterobicycloalkyl and optionallysubstituted (C₈-C₁₈)heterotricycloalkyl.

[0016] In a preferred embodiment, R¹ is a functional group including,but not limited to, optionally substituted (C₅-C₁₈)cycloalkyl or a(C₈-C₁₈)heterocycloalkyl group, more preferably a (C₈-C₁₈)bicycloalkyl,(C₈-C₁₈)tricycloalkyl, (C₈-C₁₈)heterobicycloalkyl or(C₈-C₁₈)heterotricycloalkyl group. In particularly preferredembodiments, R¹ represents an optionally substitutedtricyclo[3.3.1.1^(3,7)]decanyl (or adamantyl), bicyclo[3.2.1]octanyl,bicyclo[5.2.0]nonanyl, bicyclo[4.3.2]undecanyl,tricyclo[2.2.1.0¹]heptanyl, tricyclo[5.3.1.1¹]dodecanyl,tricyclo[5.4.0.0^(2,9)]undecanyl, tricyclo[5.3.2.0^(4,9)]dodecanyl,tricyclo[4.4.1.1^(1,5)]dodecanyl or tricyclo[5.5.1.0^(3,11)]tridecanylgroup. More preferably, R¹ is a substituted or unsubstituted adamantylgroup, most preferably an unsubstituted 1-adamantyl group.

[0017] R² is a group including, but not limited to, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted arylalkyl and optionally substitutedheteroarylalkyl. Preferred embodiments are those in which R² isaryl(C₁-C₈)alkyl or heteroaryl(C₁-C₈)alkyl. More preferably, R² isbranched heteroaryl(C₂-C₈)alkyl, for example, 1-(furan-2-yl)ethyl,1-(pyridin-2-yl)ethyl, 1-(furan-2-yl)-2-propyl, 1-(2-pyridyl)-2-propyl,1-(furanyl)isobutyl, 1-(3-pyridyl)isobutyl, 1-(pyridin-4-yl)ethyl,1-(pyridin-4-yl)isobutyl, and the like. Most preferably, R² is1-(furan-2-yl)ethyl or 1-(pyridin-2-yl)ethyl. In still other preferredembodiments, R² is a branched (C₃-C₈)alkyl, more preferably an isopropylgroup. In yet other preferred embodiments, R² is aheteroaryl(C₃-C₈)alkenyl group. More preferably, R² is a1-(3-furanyl)-3-butenyl group.

[0018] X is a functional group including, but not limited to, —CO₂R¹¹,—CH₂OR¹¹, —C(O)R¹¹, —C(O)NR¹¹R¹² and —CH₂NR¹¹R¹², wherein R¹¹ and R¹²are each members independently selected from hydrogen and optionallysubstituted (C₁-C₈)alkyl. In certain aspects, the present invention alsoprovides a combinatorial library that contains substituted benzanilides,wherein the benzanilides are optionally connected to a solid support.

[0019] In yet another aspect, the present invention provides methods forsynthesizing libraries of substituted benzanilides. having the formula:

[0020] wherein R¹ is a group including, but not limited to, optionallysubstituted (C₅-C₁₈)cycloalkyl or a (C₅-C₁₈)heterocycloalkyl group, morepreferably a (C₈-C₁₈)bicycloalkyl, (C₈-C₁₈)tricycloalkyl,(C₈-C₁₈)heterobicycloalkyl or (C₈-C₁₈)heterotricycloalkyl group. Inparticularly preferred embodiments, R¹ represents an optionallysubstituted tricyclo[3.3.1.1^(3,7)]decanyl (or adamantyl),bicyclo[3.2.1]octanyl, bicyclo[5.2.0]nonanyl, bicyclo[4.3.2]undecanyl,tricyclo[2.2.1.0¹]heptanyl, tricyclo[5.3.1.1¹]dodecanyl,tricyclo[5.4.0.0^(2,9)]undecanyl, tricyclo[5.3.2 .0^(4,9)]dodecanyl,tricyclo[4.4.1.1^(1,5)]dodecanyl or tricyclo[5.5.1.0^(3,11)]tridecanylgroup. More preferably, R¹ is a substituted or unsubstituted adamantylgroup, most preferably an unsubstituted 1-adamantyl group.

[0021] R² is a group including, but not limited to, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted arylalkyl and optionally substitutedheteroarylalkyl. Preferred embodiments are those in which R² isaryl(C₁-C₈)alkyl or heteroaryl(C₁-C₈)alkyl. More preferably, R² isbranched heteroaryl(C₂-C₈)alkyl, for example, 1-(furan-2-yl)ethyl,1-(pyridin-2-yl)ethyl, 1-(furan-2-yl)-2-propyl, 1-(2-pyridyl)-2-propyl,1-(furanyl)isobutyl, 1-(3-pyridyl)isobutyl, 1-(pyridin-4-yl)ethyl,1-(pyridin-4-yl)isobutyl, and the like. Most preferably, R² is1-(furan-2-yl)ethyl or 1-(pyridin-2-yl)ethyl. In still other preferredembodiments, R² is a branched (C₃-C₈)alkyl, more preferably an isopropylgroup. In yet other preferred embodiments, R² is aheteroaryl(C₃-C₈)alkenyl group. More preferably, R² is a1-(3-furanyl)-3-butenyl group.

[0022] X is a group including, but not limited to, —CO₂R¹¹, —CH₂OR¹¹,—C(O)R¹¹, —C(O)NR¹¹R¹² and —CH₂NR¹¹R¹², wherein R¹¹ and R¹² are eachmembers independently selected from hydrogen and optionally substituted(C₁-C₈)alkyl. In certain aspects, the present invention also provides acombinatorial library that contains substituted benzanilides, whereinthe benzanilides are optionally connected to a solid support.

[0023] The present invention also provides a method for screening alibrary that contains a substituted benzanilide of Formula I optionallyconnected to a solid support.

[0024] These and other features and advantages will become more apparentwhen read with the accompanying drawings and detailed description thatfollow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0025]FIG. 1 illustrates general synthetic routes to substitutedbenzanilides using solid support technology;

[0026]FIG. 2 illustrates various embodiments of coupling, cleavageconditions, and products formed using solid support technology of thepresent invention; and

[0027]FIG. 3 illustrates a purification embodiment using solid supporttechnology of the present invention.

DEFINITIONS

[0028] As used herein, “chemical library” or “array” is an intentionallycreated collection of differing molecules which can be preparedsynthetically and screened for biological activity in a variety ofdifferent formats (e.g., libraries of soluble molecules, libraries ofmolecules bound to a solid support).

[0029] The term “alkyl,” by itself or as part of another substituent,means, unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or polyunsaturated and can include di- andmulti-radicals, having the number of carbon atoms designated (i.e.C₁-C₁₀ means one to ten carbons). Examples of saturated hydrocarbonradicals include groups such as methyl, ethyl, n-propyl, isopropyl,n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl,cyclopropylmethyl, homologs and isomers of, for example, n-pentyl,n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group isone having one or more double bonds or triple bonds. Examples ofunsaturated alkyl groups include vinyl, 2-propenyl, crotyl,2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl),ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs andisomers. The term “alkyl,” unless otherwise noted, is also meant toinclude those derivatives of alkyl defined in more detail below as“cycloalkyl” and “alkylene.” The term “alkylene” by itself or as part ofanother substituent means a divalent radical derived from an alkane, asexemplified by —CH₂CH₂CH₂CH₂—. Typically, an alkyl group will have from1 to 24 carbon atoms, with those groups having 10 or fewer carbon atomsbeing preferred in the present invention. A “lower alkyl” or “loweralkylene” is a shorter chain alkyl or alkylene group, generally havingeight or fewer carbon atoms.

[0030] The term “alkoxy,” employed alone or in combination with otherterms means, unless otherwise stated, an alkyl group, as defined above,connected to the remainder of the molecule via an oxygen atom, such as,for example, methoxy, ethoxy, 1-propoxy, 2-propoxy and the higherhomologs and isomers.

[0031] The term “heteroalkyl,” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and from one to threeheteroatoms selected from the group consisting of O, N, Si and S, andwherein the nitrogen and sulfur atoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized. The heteroatom(s) O,N and S may be placed at any interior position of the heteroalkyl group.The heteroatom Si may be placed at any position of the heteroalkylgroup, including the position at which the alkyl group is attached tothe remainder of the molecule. Examples include —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Also included in the term“heteroalkyl” are those radicals described in more detail below as“heteroalkylene” and “heterocycloalkyl.” The term “heteroalkylene” byitself or as part of another substituent means a divalent radicalderived from heteroalkyl, as exemplified by —CH₂—CH₂—S—CH₂CH₂— and—CH₂—S—CH₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini. Still further, for alkyleneand heteroalkylene linking groups, as well as all other linking groupsdescribed herein, no specific orientation of the linking group isimplied.

[0032] The terms “cycloalkyl” and “heterocycloalkyl”, by themselves orin combination with other terms, represent, unless otherwise stated,cyclic versions of “alkyl” and “heteroalkyl”, respectively. The terms“cycloalkyl” and “heterocycloalkyl” are also meant to include bicyclic,tricyclic and polycyclic versions thereof. Additionally, forheterocycloalkyl, a heteroatom can occupy the position at which theheterocycle is attached to the remainder of the molecule. Examples ofcycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl,3-cyclohexenyl, cycloheptyl, bicyclo[2.2.1]heptyl, bicyclo[2.2.2]octyl,adamantyl, and the like. Examples of heterocycloalkyl include1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl,3-piperidinyl, 4-morpholinyl, 3-morpholinyl,1,4-diazabicyclo[2.2.2]oct-2-yl, tetrahydrofuran-2-yl,tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl,1-piperazinyl, 2-piperazinyl, and the like.

[0033] The terms “halo” or “halogen,” by themselves or as part ofanother substituent, mean, unless otherwise stated, a fluorine,chlorine, bromine, or iodine atom. Additionally, terms such as“fluoroalkyl,” are meant to include monofluoroalkyl and polyfluoroalkyl.

[0034] The term “aryl,” employed alone or in combination with otherterms (e.g., aryloxy, arylthioxy, arylalkyl) means, unless otherwisestated, an aromatic substituent which can be a single ring or multiplerings (up to three rings) which are fused together or linked covalently.The rings may each contain from zero to four heteroatoms selected fromN, O, and S, wherein the nitrogen and sulfur atoms are optionallyoxidized, and the nitrogen atom(s) are optionally quaternized. The arylgroups that contain heteroatoms may be referred to as “heteroaryl” andcan be attached to the remainder of the molecule through a carbon atomor a heteroatom. Non-limiting examples of aryl groups include phenyl,1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl,3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl,4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl,2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl,3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl,5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl,5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and6-quinolyl. Substituents for each of the above noted aryl ring systemsare selected from the group of acceptable substituents described below.

[0035] The term “acyl” denotes the —C(O)R group, wherein R is alkyl oraryl as defined above, such as formyl, acetyl, propionyl, or butyryl.

[0036] The term “acyl halide” denotes the RC(O)X group, wherein R isalkyl or aryl as defined above, and X is a halogen.

[0037] The terms “arylalkyl” and “arylheteroalkyl” are meant to includethose radicals in which an aryl group is attached to an alkyl group(e.g., benzyl, phenethyl, pyridylmethyl and the like) or a heteroalkylgroup (e.g., phenoxymethyl, 2-pyridyloxymethyl, 1-naphthyloxy-3-propyl,and the like). The arylalkyl and arylheteroalkyl groups will typicallycontain from 1 to 3 aryl moieties attached to the alkyl or heteroalkylportion by a covalent bond or by fusing the ring to, for example, acycloalkyl or heterocycloalkyl group. For arylheteroalkyl groups, aheteroatom can occupy the position at which the group is attached to theremainder of the molecule. For example, the term “arylheteroalkyl” ismeant to include benzyloxy, 2-phenylethoxy, phenethylamine, and thelike.

[0038] Each of the above terms (e.g., “alkyl,” “heteroalkyl” and “aryl”)are meant to include both substituted and unsubstituted forms of theindicated radical. Preferred substituents for each type of radical areprovided below.

[0039] Substituents for the alkyl and heteroalkyl radicals (includingthose groups often referred to as alkylene, alkenyl, heteroalkylene,heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, andheterocycloalkenyl) can be a variety of groups selected from: —OR′, ═O,═NR′, ═N—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —CO₂R′,—CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′, —NH—C(NH₂)═NH,—NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —CN and—NO₂ in a number ranging from zero to (2N+1), where N is the totalnumber of carbon atoms in such radical. R′, R″ and R′″ eachindependently refer to hydrogen, unsubstituted(C₁-C₈)alkyl andheteroalkyl, unsubstituted aryl, aryl substituted with 1-3 halogens,unsubstituted alkyl, alkoxy or thioalkoxy groups, or aryl-(C₁-C₄)alkylgroups. When R′ and R″ are attached to the same nitrogen atom, they canbe combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include 1-pyrrolidinyl and4-morpholinyl.

[0040] Similarly, substituents for the aryl groups are varied and areselected from: -halogen, —OR′, —OC(O)R′, —NR′R″, —SR′, —R′, —CN, —NO₂,—CO₂R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR″C(O)₂R′, —NH—C(NH₂)═NH,—NR′C(NH₂)═NH, —NH—C(NH₂)═NR′, —S(O)R′, —S(O)₂R′, —S(O)₂NR′R″, —N₃,—CH(Ph)₂, perfluoro(C₁-C₄)alkoxy, and perfluoro(C₁-C₄)alkyl, in a numberranging from zero to the total number of open valences on the aromaticring system; and where R′ and R″ are independently selected fromhydrogen, (C₁-C₈)alkyl and heteroalkyl, unsubstituted aryl,(unsubstituted aryl)-(C₁-C₄)alkyl, and (unsubstitutedaryl)oxy-(C₁-C₄)alkyl.

[0041] Two of the substituents on adjacent atoms of the aryl ring mayoptionally be replaced with a substituent of the formula-T-C(O)′(CH₂)_(q)—U—, wherein T and U are independently —NH—, —O—, —CH₂—or a single bond, and q is an integer of from 0 to 2. Alternatively, twoof the substituents on adjacent atoms of the aryl ring may optionally bereplaced with a substituent of the formula -A-(CH₂)_(r)—B—, wherein Aand B are independently —CH₂—, —O—, —NH—, —S—, —S(O)—, —S(O)₂—,—S(O)₂NR′— or a single bond, and r is an integer of from 1 to 3. One ofthe single bonds of the new ring so formed may optionally be replacedwith a double bond. Alternatively, two of the substituents on adjacentatoms of the aryl ring may optionally be replaced with a substituent ofthe formula —(CH₂)_(s)—X—(CH₂)_(t)—, where s and t are independentlyintegers of from 0 to 3, and X is —O—, —NR′—, —S—, —S(O)—, —S(O)₂—, or—S(O)₂NR′—. The substituent R′ in —NR′— and —S(O)₂NR′— is selected fromhydrogen or unsubstituted (C₁-C₆)alkyl.

[0042] As used herein, the term “heteroatom” is meant to include oxygen(O), nitrogen (N), sulfur (S) and silicon (Si).

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

[0043] A. Methods for Preparing Modulators of LXRs

[0044] The present invention provides methods for solid phase synthesisof modulators of LXRs. The methods are useful for producingcombinatorial libraries of modulators of LXRs. The modulators of LXRscan be antagonists and/or agonists. As such, the present inventionprovides a method for preparing LXR ligands on a solid support,comprising:

[0045] (a) attaching an aniline derivative to the solid support toprovide a support-bound aniline derivative;

[0046] (b) contacting the support-bound aniline derivative with analdehyde or ketone under reductively aminating conditions to provide asupport-bound substituted aniline derivative; and

[0047] (c) contacting the support-bound substituted aniline derivativewith an acylating agent to provide an LXR ligand on the solid support.

[0048] Various solid support materials are amenable to the methodsdescribed herein. Suitable solid supports include, but are not limitedto, agarose, polyacrylamide, polystyrene, polyacrylate,hydroxethylmethacrylate, polyamide, polyethylene, polyethyleneoxy, orcopolymers and grafts of such. Other embodiments of solid-supportsinclude small particles, non-porous surfaces, addressable arrays, etc.In certain aspects, the solid supports include, but are not limited to,polymer resins (e.g., polyethylene glycol and polystyrene), gels (e.g.,polyethylene glycol gels), polyacrylamide/polyethylene glycol copolymerresins, controlled pore glass supports (e.g., the CPG supportscommercially available from Millipore), and silica beads and wafers.

[0049] In certain embodiments, the solid support of the presentinvention has a “linker” group attached thereto that serves to appendthe growing modulator or “target molecule” to the solid support.Preferably, the linker is the point of cleavage following synthesis.Thus, it is advantageous that the linkers be labile to particular acidor base conditions. In certain other embodiments, the linkers arephotolabile. Preferred linkers include, but are not limited to,4-(bromomethyl)-phenoxymethyl polystyrene (e.g., Wang resin), Merrifieldresin, Rink amide resin and Sieber resin. In one preferred embodiment,the solid support-bound linker is4-[2′,4′-dimethoxyphenyl)-aminomethyl]-phenoxymethyl-linked polystyreneresin (“Rink's amide resin”).

[0050]FIG. 1 illustrates one particular embodiment of the presentinvention. FIG. 1 is merely an example that should not limit the scopeof the claims herein. One of ordinary skill in the art will recognizemany other variations, alternatives, and modifications. In certainembodiments, the aniline derivative to be attached to the solid supportis first protected with a suitable protecting group. As used herein,“PG” or “protecting group” refers to a chemical group that exhibits thefollowing characteristics: (1) reacts selectively with the desiredfunctionality in good yield to give a protected substrate that is stableto the projected reactions for which protection is desired; 2) isselectively removable from the protected substrate to yield the desiredfunctionality; and 3) is removable in good yield by reagents compatiblewith the other functional group(s) generated in suc h protectedreactions. Examples of protecting groups can be found in Greene et al.(1991) Protective Groups in Organic Synthesis, 2nd Ed. (John Wiley &Sons, Inc., New York). A preferred protecting group is9-fluorenylmethyloxycarbonyl or Fmoc. The Fmoc group protects theprimary amine. Other protecting groups are well known to those of skillin the art.

[0051] In certain aspects, the aniline derivatives of the presentinvention have the formula:

[0052] Various solvent systems can be used in the methods of the presentinvention. For the coupling of the aniline derivative to the linkergroup, an anhydrous solvent system is preferred. For example, when theWang resin is used, coupling conditions involve for example,diisopropylethylamine (DIEA)/CsI in DMF. For use of the Rink's amideresin, the coupling conditions involve for example,benzotriazol-1-yl-oxytris(pyrrolidino)phosphonium hexafluorophosphate(PyBop)/DIEA in DMF. Those of skill in the art will know of othersolvent systems and coupling reagents suitable for use in the presentinvention.

[0053] After the aniline derivative is coupled to the linker (step 1),the protecting group can be displaced. Various deprotection systems arewell known in the art. Standard deprotection conditions include, forexample, a solution comprising 20% piperidine in DMF. Those of skill inthe art will know of other deprotection schemes suitable for use in thepresent invention.

[0054] In certain aspects, the aniline derivative that is attached tothe solid support is alkylated through a reductive amination reactionwith an aldehyde or a ketone using a reducing agent. The process can bestepwise (i.e., isolation of a resin-bound derivative followed byreduction) or direct (i.e., addition of carbonyl compound i.e., analdehyde or ketone, and reduction in the same step). By contacting thesupport-bound aniline derivative with an aldehyde or ketone underreductively aminating conditions, the methods of the present inventionprovide a support-bound substituted aniline derivative (step 2). As usedherein, the phrase “reductive amination conditions” refers to a reactionof an aldehyde or a ketone with a primary amine in the presence ofreducing agent to yield a secondary amine. As shown in FIG. 1, thealdehyde or ketone is reductively aminated, while the primary amine isconcomitantly reductively alkylated (step 2).

[0055] A variety of reducing agents are suitable for use in the methodsof the present invention. Suitable reducing agents include, but are notlimited to, sodium cyanoborohydride, sodium triacetoxyborohydride sodiumborohydride, lithium aluminum hydride, and borane. In a preferredembodiment, sodium cyanoborohydride is used.

[0056] Various aldehydes and ketones can be used in the methods of thepresent invention. The aldehyde or ketone is added to the reactionmixture under reductively aminating conditions to provide asupport-bound substituted aniline derivative. Suitable aldehydes includealiphatic aldehydes, aromatic aldehydes and mixtures thereof. Suitablealdehydes include, but are not limiting to, acetaldehyde,propionaldehyde, buryraldehyde, pentanal, hexanal, etc. and aromaticaldehydes including but not limited to, benzaldehyde, 1-naphthaldehyde,4-acetoxybenzaldehyde, 4-benzoyloxybenzaldehyde, etc.

[0057] Likewise, a wide variety of ketones can be reductively aminatedusing the methods of the present invention. Suitable ketones includealiphatic ketones, aromatic ketones and mixtures thereof. Suitableketones include, but are not limited to, 2-propanone,1-(3,4-dimethoxyphenyl)-2-propanone, cyclohexanone, cyclopentanone,3-methyl-2-butanone, 4-hydroxy-2-pentanone,4-(4-hydroxyphenyl)-2-butanone, 2-methoxyacetone,4,4-dimethyl-2-pentanone, diphenyl ketone, etc.

[0058] In certain instances, the ketones suitable for use in the methodsof the present invention have the formula:

R³—C(O)—R⁴

[0059] wherein R³ and R⁴ are members each independently selected fromoptionally substituted aryl, optionally substituted heteroaryl,optionally substituted arylalkyl, optionally substitutedheteroarylalkyl, and optionally substituted alkyl.

[0060] With reference to FIG. 1, after the carbonyl compound has beenreductively aminated, an acylating reagent is thereafter added (step 3).Suitable acylating reagents include, but are not limited to, “acidhalides” (e.g., acid chlorides like acetyl chloride and propionylchloride, acid fluorides, and acid bromides) and “acid anhydrides”(e.g., acetic anhydride and formic acetic anhydride). The term “acidanhydride” refers to symmetrical, asymmetrical, and mixed anhydrides.The terms “acylation,” “acylating,” and the like, refer to a chemicalreaction whereby an acyl group is added to another moiety.

[0061] In the present methods, an acyl group is added to the substitutedaniline derivative to provide an LXR ligand on a solid support.Preferred acylating agents include, but are not limited to, a carboxylicacid, a carboxylate ester, a carboxylic acid halide and other activatedforms of carboxylic acids, such as a reactive anhydride. Reactive acidhalides include for example, acid chlorides, acid bromides, and acidfluorides. Preferred acid halides are acid chlorides.

[0062] In certain preferred methods, the acylating agents of the presentinvention have the formula

R¹—Y

[0063] wherein: R¹ is selected from optionally substituted(C₈-C₁₈)bicycloalkyl, optionally substituted (C₈-C₁₈)tricycloalkyl,optionally substituted (C₈-C₁₈)heterobicycloalkyl and optionallysubstituted (C₈-C₁₈)heterotricycloalkyl; and Y is selected from acarboxylic acid, a carboxylate ester, a carboxylic acid chloride andother activated forms of carboxylic acids. In a preferred embodiment,R¹—Y is 1-adamantanecarbonyl chloride.

[0064] Optionally, the LXR ligand is removed from the solid supportusing condition well known to those of skill in the art (step 4).General mild cleavage conditions include for example, trifluoroaceticacid in water.

[0065]FIG. 2 illustrates one particular embodiment of the presentinvention. FIG. 2 is merely an example that should not limit the scopeof the claims herein. One of ordinary skill in the art will recognizemany other variations, alternatives, and modifications. In certaininstances, the reagents used to cleave the molecule on the solid supportwill generate products having different functionalities. After theaminated product is acylated, various cleavage reagents can be used togenerate an LXR modulator. Table 1 below shows various coupling/cleavingconditions and products associated with those conditions. TABLE 1 ResinCoupling Conditions Cleavage Conditions Product Wang or DIEA/CsI/DMF TFA—CO₂H Merrifield DIBAL/toluene —CH₂OH NaOMe/MeOH —CO₂Me Rink amide resinPyBOP/DIEA/DMF TFA —CONH₂ or Sieber

[0066] B. Combinatorial Synthesis

[0067] Parallel, or combinatorial synthesis has as its primary objectivethe generation of a library of diverse molecules which all share acommon feature. By substituting different moieties at each of thevariable parts of the scaffold molecule, the amount of space explorablein a library grows. Theories and modern medicinal chemistry advocate theconcept of occupied space as a key factor in determining the efficacy ofa given compound against a given biological target. By creating adiverse library of molecules that explores a large percentage of thetargeted space, the odds of developing a highly efficacious leadcompound increase dramatically.

[0068] Chemical combinatorial libraries are diverse collections ofmolecular compounds (see, Gordon et al. (1995) Acc. Chem. Res.29:144-154). These compounds are formed using a multi step syntheticroute, wherein a series of different chemical modules can be inserted atany particular step in the route. By performing the synthetic routemultiple times in parallel, each possible permutation of the chemicalmodules can be constructed. The result is the rapid synthesis ofhundreds, thousands, or even millions of different structures within achemical class.

[0069] The various elements of the library are typically varied by, forexample, the parallel dispensing of reagents to spatially addressablesites or by known “split-and-pool” combinatorial methodology. Othertechniques for assembling combinatorial libraries will be apparent tothose of skill in the art. See, for example, Thompson, L. A., et al.,“Synthesis and Applications of Small Molecule Libraries,” Chem. Rev.1996, 96:555-600, and references therein which are herein incorporatedby reference. See also, for example, Kaldor et al., “Synthetic OrganicChemistry on Solid Support” In, COMBINATORIAL CHEMISTRY AND MOLECULARDIVERSITY IN DRUG DISCOVERY, Gordon et al., Eds., Wiley-Liss, New York,1998.

[0070] Parallel synthesis of “small” molecules (non-oligomers with amolecular weight of 200-1000) was rarely attempted prior to 1990. See,for example, Camps. et al., Annaks de Quimica, 70: 848 (1990). Recently,Ellmann disclosed the solid phase-supported parallel (also referred toas “combinatorial”) synthesis of eleven benzodiazepine analogs alongwith some prostaglandins and beta-turn mimetics. These disclosures areexemplified in U.S. Pat. No. 5,288,514. Another relevant disclosure ofparallel synthesis of small molecules may be found in U.S. Pat. No.5,324,483. This patent discloses the parallel synthesis of between 4 and40 compounds in each of sixteen different scaffolds. Chen et al. havealso applied organic synthetic strategies to develop non-peptidelibraries synthesized using multi-step processes on a polymer support.(Chen et al., J. Am. Chem. Soc., 116: 2661-2662 (1994)).

[0071] In one embodiment, the present invention provides a method ofsolid phase synthesis to generate a chemical library of single compoundsthat bind to the LXRα receptor using the Spatially Determined Arraymethod. In this method, protected amines are independently attached tothe solid support (see, FIG. 1, step 1) and maintained in separateflasks. After synthesis is complete, the final product is then releasedfrom the solid support through a cleavage procedure (see, FIG. 1, step4; and Table 1). This method saves the time and effort spent in compoundisolation and compound purification using the tradition solution phasemethods, that often involve the isolation and purification ofintermediates and final products.

[0072] In another embodiment of the present invention, a large number ofsolid support beads or particles are suspended in a suitable carrier(such as a solvent) in a parent container. The beads, for example, areprovided with a functionalized point of attachment (“the linker”) for achemical module. The beads are then divided and placed in variousseparate reaction vessels. The first chemical module (“the anilinederivative”) is attached to the bead, providing a variety of differentlysubstituted solid supports. Where the first chemical module includes 3different members, the resulting substituted beads can be represented asA1, A2, and A3.

[0073] Thereafter, the beads are washed to remove excess reagents andsubsequently remixed in the parent container. The bead mixture is againdivided and placed into various separate reaction vessels. The secondchemical module (“the aldehyde or ketone”) is coupled to the firstchemical module. Where the second chemical module includes 3 differentmembers, B1, B2, and B3, 9 differently substituted beads result: A1 B1,A1 B2, A1 B3, A2B1, A2B2, A2B3, A3B1, A3B2, and A3B3. Each bead willhave only a single type of molecule attached to its surface.

[0074] The remixing/redivision synthetic process can be repeated untileach of the different chemical modules has been incorporated into themolecule attached to the solid support. Through this method, largenumbers of individual compounds can be rapidly and efficientlysynthesized. For instance, where there are 3 different chemical modules,and where each chemical module contains 20 members, 8000 beads ofdifferent molecular substitution can be produced.

[0075] In general, combinatorial library synthesis can be performedeither manually or through the use of an automated process. For themanual construction of a combinatorial library, a scientist wouldperform the various chemical manipulations. For the construction of acombinatorial library through an automated process, the various chemicalmanipulations will typically be performed robotically. Robotics used inconjunction with high throughput screening methods have only recentlyexperienced general use. Development of robotics systems that can handlelarge numbers of resin samples for proportioning, mixing, cleavage andsample handling will continue to be developed. Moreover, robotics thatcan perform multiple chemical reactions at variable temperatures, andsubsequently handle work up and spectroscopic characterization ofbioactive leads, are or will become generally available. Manipulation oflibraries containing billions of compounds will provide an impetus toimprove resin loading and handling capabilities. With the availabilityof new assay formats, partially cleavable libraries can beadvantageously employed, and solid phase assays to identify enzymeinhibitors and ligands for soluble receptors will become available.Additional selection means that enable identification of activecompounds within enormous combinatorial libraries can feature affinityenrichment or affinity selection, followed by mass spectroscopicidentification of any bioactive compound.

[0076] As such, the present invention provides a combinatorial librarycomprising compounds having the formula:

[0077] wherein R¹ is a group including, but not limited to, optionallysubstituted (C₅-C₁₈)cycloalkyl or a (C₅-C₁₈)heterocycloalkyl group, morepreferably a (C₈-C₁₈)bicycloalkyl, (C₈-C₁₈)tricycloalkyl,(C₈-C₁₈)heterobicycloalkyl or (C₈-C₁₈)heterotricycloalkyl group. Inparticularly preferred embodiments, R¹ represents an optionallysubstituted tricyclo[3.3.1.1^(3,7)]decanyl (or adamantyl),bicyclo[3.2.1]octanyl, bicyclo[5.2.0]nonanyl, bicyclo[4.3.2]undecanyl,tricyclo[2.2.1.0¹]heptanyl, tricyclo[5.3.1.1¹]dodecanyl,tricyclo[5.4.0.0^(2,9)]undecanyl, tricyclo[5.3.2.0^(4,9)]dodecanyl,tricyclo[4.4.1.1^(1,5)]dodecanyl or tricyclo[5.5.1.0^(3,11)]tridecanylgroup. More preferably, R¹ is a substituted or unsubstituted adamantylgroup, most preferably an unsubstituted 1-adamantyl group.

[0078] R² is a group including, but not limited to, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted arylalkyl and optionally substitutedheteroarylalkyl. Preferred embodiments are those in which R² isaryl(C₁-C₈)alkyl or heteroaryl(C₁-C₈)alkyl. More preferably, R² isbranched heteroaryl(C₂-C₈)alkyl, for example, 1-(furan-2-yl)ethyl,1-(pyridin-2-yl)ethyl, 1-(furan-2-yl)-2-propyl, 1-(2-pyridyl)-2-propyl,1-(furanyl)isobutyl, 1-(3-pyridyl)isobutyl, 1-(pyridin-4-yl)ethyl,1-(pyridin-4-yl)isobutyl, and the like. Most preferably, R² is1-(furan-2-yl)ethyl or 1-(pyridin-2-yl)ethyl. In still other preferredembodiments, R² is a branched (C₃-C₈)alkyl, more preferably an isopropylgroup. In yet other preferred embodiments, R² is aheteroaryl(C₃-C₈)alkenyl group. More preferably, R² is a1-(3-furanyl)-3-butenyl group.

[0079] X is a group including, but not limited to, —CO₂R¹¹, —CH₂OR¹¹,—C(O)R¹¹, —C(O)NR¹¹R¹² and —CH₂NR¹¹R¹², wherein R¹¹ and R¹² are eachmembers independently selected from hydrogen and optionally substituted(C₁-C₈)alkyl. In certain aspects, the present invention also provides acombinatorial library that contains substituted benzanilides, whereinthe benzanilides are optionally connected to a solid support.

[0080] In another aspect, the present invention provides methods forsynthesizing libraries of substituted benzanilides having the formula:

[0081] R¹ is a group including, but not limited to, optionallysubstituted (C₅-C₁₈)cycloalkyl or a (C₅-C₁₈)heterocycloalkyl group, morepreferably a (C₈-C₁₈)bicycloalkyl, (C₈-C₁₈)tricycloalkyl,(C₈-C₁₈)heterobicycloalkyl or (C₈-C₁₈)heterotricycloalkyl group. Inparticularly preferred embodiments, R¹ represents an optionallysubstituted tricyclo[3.3.1.1^(3,7)]decanyl (or adamantyl),bicyclo[3.2.1]octanyl, bicyclo[5.2.0]nonanyl, bicyclo[4.3.2]undecanyl,tricyclo[2.2.1.0¹]heptanyl, tricyclo[5.3.1.1¹]dodecanyl,tricyclo[5.4.0.0^(2,9)]undecanyl, tricyclo[5.3.2.0^(4,9)]dodecanyl,tricyclo[4.4.1.1^(1,5)]dodecanyl or tricyclo[5.5.1.0^(3,11)]tridecanylgroup. More preferably, R¹ is a substituted or unsubstituted adamantylgroup, most preferably an unsubstituted 1-adamantyl group.

[0082] R² is a group including, but not limited to, optionallysubstituted alkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted arylalkyl and optionally substitutedheteroarylalkyl. Preferred embodiments are those in which R² isaryl(C₁-C₈)alkyl or heteroaryl(C₁-C₈)alkyl. More preferably, R² isbranched heteroaryl(C₂-C₈)alkyl, for example, 1-(furan-2-yl)ethyl,1-(pyridin-2-yl)ethyl, 1-(furan-2-yl)-2-propyl, 1-(2-pyridyl)-2-propyl,1-(furanyl)isobutyl, 1-(3-pyridyl)isobutyl, 1-(pyridin-4-yl)ethyl,1-(pyridin-4-yl)isobutyl, and the like. Most preferably, R² is1-(furan-2-yl)ethyl or 1-(pyridin-2-yl)ethyl. In still other preferredembodiments, R² is a branched (C₃-C₈)alkyl, more preferably an isopropylgroup. In yet other preferred embodiments, R² is aheteroaryl(C₃-C₈)alkenyl group. More preferably, R² is a1-(3-furanyl)-3-butenyl group.

[0083] X is a group including, but not limited to, —CO₂R¹¹, —CH₂OR¹¹,—C(O)R¹¹, —C(O)NR¹¹R¹² and —CH₂NR¹¹R¹², wherein R¹¹ and R¹² are eachmembers independently selected from hydrogen and optionally substituted(C₁-C₈)alkyl. In certain aspects, the present invention also provides acombinatorial library that contains substituted benzanilides, whereinthe benzanilides are optionally connected to a solid support.

[0084] The compounds of Formula I can be purified using the scheme setforth in FIG. 3, which is merely an example that should not limit thescope of the claims herein. One of ordinary skill in the art willrecognize many other variations, alternatives, and modifications.

[0085] C. Screening

[0086] Screening technologies are undergoing, and will continue toundergo, considerable changes and improvements, especially with regardto functional screening techniques. One aim is development of simple andrapid functional assays that can identify one or more active ingredientsin tested pools without the need for a long deconvolution process.Substantial improvements in chemistry and assay techniques will be keyto achieving generalized, routine use of such assays. For instance, thescope and versatility of organic reactions conducted on solid supportsare increasing. Such improvements will enrich methods and diversityrelating to small molecule combinatorial libraries.

[0087] Methods for isolating library compound species that demonstratedesirable affinity for a receptor such as a LXRs or enzymes are wellknown in the art. For example, a receptor solution may be mixed with asolution of the compounds of a particular combinatorial library underconditions favorable to receptor-ligand binding. Specific binding oflibrary compounds to the receptor can be detected by any of the numerousreceptor bindingassays that are well known in the art. Compounds thatare bound to the receptor can be readily separated from compounds thatremain free in solution by applying the solution to a Sephadex G-25 gelfiltration column. Free receptor and receptor-ligand complexes will passthrough the column quickly, while free library compounds will beretarded in their progress through the column. The mixture ofreceptor-ligand complex and free receptor can then be treated with apowerful denaturing agent, such as guanidinium hydrochloride or urea, tocause release of the ligand from the receptor. The solution can then beinjected onto an HPLC column (for example, a Vydac C-4 reverse-phasecolumn, eluted with a gradient of water and acetonitrile ranging from 0%acetonitrile to 80% acetonitrile). Diode array detection can providediscrimination of the compounds of the combinatorial library from thereceptor. The compound peaks can then collected and subjected to massspectrometry for identification.

[0088] Finding a compound that inhibits an enzyme or binds to a receptoris most readily performed with free compound in solution. The compoundscan also be screened while still bound to the resin used for synthesis.In some applications, this may be the preferable mode of findingcompounds with the desired characteristics. For example, if a compoundthat binds to a specific ligand is desired, the resin-bound library ofcompounds can be contacted with a ligand in solution under conditionsfavoring a stable ligand-compound-resin complex. The bead can then bephysically removed from the resin mixture and subjected to mass spectralanalysis. If the synthesis has been conducted in a manner such that onlyone compound is likely to be synthesized on a particular bead, then thebinding compound has been identified. If the synthesis has been carriedout so that many compounds are present on a single bead, the informationderived from analysis can be utilized to narrow the synthetic choicesfor the next round of synthesis and identification.

[0089] The enzyme or receptor target need not be in solution either. Thereceptor or enzyme may be immobilized on a column. The library ofcompounds may then be passed over the column, resulting in the retentionof strongly binding compounds on the column after weaker-binding andnon-binding compounds are washed away. The column can then be washedunder conditions that dissociate protein-ligand binding, which willremove the compounds retained in the initial step. These compounds canthen be analyzed, and synthesized separately in quantity for furthertesting. Similarly, cells bearing surface receptors can be expressed ona cell surface may be contacted with a solution of library compounds.The cells bearing bound compounds can be readily separated from thesolution containing non-binding compounds. The cells can then be washedwith a solution that will dissociate the bound ligand from the cellsurface receptor. Again, the cells can be separated from the solution,and the solution that now contains the ligands bound in the initial stepcan be analyzed.

[0090] Compounds contained within the combinatorial libraries of thepresent invention, are capable of specifically modulating LXRs, forexample regulating LXRα. Compounds may be evaluated in vitro for theirability to activate LXRα receptor function using cell-based assays suchas that described in Lehmann, et al. (J. Biol. Chem. 1997, 272(6),3137-3140) or biochemical assays (see co-pending applications Ser. No.08/975,614 (filed Nov. 21, 1997) and Ser. No. 09/163,713 (filed Sep. 30,1998)). Alternatively, the libraries can be evaluated for their abilityto increase or decrease gene expression modulated by LXR, usingwestern-blot analysis. Established animal models to evaluatehypocholesterolemia effects of compounds are known in the art. Forexample, compounds disclosed herein can be tested for their ability tolower cholesterol levels in hamsters fed a high-cholesterol diet, usinga protocol similar to that described in Spady et al. (J. Clin. Invest.1988, 81, 300), Evans et al. (J. Lipid Res. 1994, 35, 1634), and Lin etal (J. Med. Chem. 1995, 38, 277). Still further, LXRα animal models(e.g., LXRα (+/−) and (−/−) mice) can be used for evaluation of thepresent compounds and compositions (see, for example, Peet, et al. Cell1998, 93, 693-704).

[0091] The following example is provided to illustrate, but not limit,the invention.

EXAMPLE

[0092] Methods

[0093]¹H-NMR spectra are recorded on a Varian Gemini 400 MHz NMRspectrometer. Significant peaks are tabulated in the order: number ofprotons, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet;m, multiplet; br s, broad singlet) and coupling constant(s) in Hertz.Electron Ionization (EI) mass spectra are recorded on a Hewlett Packard5989A mass spectrometer. Mass spectrometry results are reported as theratio of mass over charge, followed by the relative abundance of eachion.

[0094] Unless otherwise indicated, chemicals are obtained from SigmaChemical Co., St. Louis, Mo. or from Aldrich Chemical Co., Inc.,Milwaukee, Wis.

[0095] Combinatorial Library

[0096] 1. Resins: Fourteen resin samples i.e., Fmoc-protectedbenzanilide derivatives are prepared and are attached to Wang resin.These resin samples are combined in a silanized beaker and mixed withDIEA/CsI and dimethyl formamide (DMF) for about 2 h. The resin samplemixture is filtered, then rinsed with DMF. The resin is then vacuumdried overnight.

[0097] 2. The deprotection step proceeds with an initial washing of theresin samples with 20% piperidine in DMF for 3 min. This step removesany residual DMF from the coupling step. The actual deprotection iscarried out for 20 additional min using 20% piperidine in DMF. Thereaction vessels are all emptied and washed prior to the reductiveamination step.

[0098] 3. Reductive deamination: In a reaction vessel is placed adeprotected benzanilide derivative and benzaldehyde. To the reactionvessel is added a solution of NaCNBH₄.

[0099] 4. Acylation reaction: The reagents contained in the vessels arethereafter acylated with benzoyl chloride.

[0100] 5. Cleavage procedure: The compound mixtures are cleaved from theresin using 95% TFA (trifluoroacetic acid)/5% water into 3 ml vials.Aliquots (1.5 mL) of the cleaved solution are added to each well andmixed for 1.5 h. After emptying each reaction vessel, the resin isrinsed with another 1.0 mL of TFA cleavage solution. The TFA is removedby directing a dry nitrogen stream over the sample vials and placing thesamples into a vacuum desiccator overnight.

[0101] 6. Screening results: Individual samples are subjected to highthroughput screening for a determination of biological LXR activity.

[0102] In order to identify agonists for LXRα, a cell-based highthroughput screen was developed. Briefly, a DNA-binding domain of thenonreceptor transcription factor GAL4 was fused to the putativeligand-binding domain of LXRα. The resulting construct is introducedinto 293 cells, together with an UAS-containing luciferase reporterconstruct. The transfected cells are then treated with the compounds andluciferase activity is measured. Compound libraries are evaluatedrelative to a control at a concentration of 10 μM. Luciferase activityis determined.

[0103] While the present invention has been described with reference tothe specific embodiments thereof, it should be understood by thoseskilled in the art that various changes may be made and equivalents maybe substituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular reaction, material, library, process, process step or steps,to the objective, spirit and scope of the present invention. All suchmodifications are intended to be within the scope of the claims appendedhereto. All publications, patents and patent applications mentioned inthis specification are herein incorporated by reference into thespecification in their entirety for all purposes.

What is claimed is:
 1. A method for preparing LXR ligands on a solidsupport, said method comprising: (a) attaching an aniline derivative tosaid solid support to provide a support-bound aniline derivative; (b)contacting said support-bound aniline derivative with an aldehyde orketone under reductively aminating conditions to provide a support-boundsubstituted aniline derivative; and (c) contacting said support-boundsubstituted aniline derivative with an acylating agent to provide an LXRligand on said solid support.
 2. A method in accordance with claim 1,further comprising: (d) removing said LXR ligand from said solidsupport.
 3. A method in accordance with claim 1, wherein said anilinederivative has the formula:

wherein PG is a protecting group, and said method further comprises astep between steps (a) and (b) of removing said protecting group.
 4. Amethod in accordance with claim 1, wherein said aldehyde or ketone ofstep (b) is selected from the group consisting of an optionallysubstituted (C₁-C₈)alkyl aldehyde and an optionally substituteddialkylketone.
 5. A method in accordance with claim 1, wherein saidaldehyde or ketone of step (b) is selected from the group consisting ofoptionally substituted aryl aldehyde and a ketone having the formulaR³—C(O)—R⁴ wherein R³ and R⁴ are members each independently selectedform the group consisting of optionally substituted aryl, optionallysubstituted heteroaryl, optionally substituted arylalkyl, optionallysubstituted heteroarylalkyl and optionally substituted alkyl.
 6. Amethod in accordance with claim 1, wherein said acylating agent has theformula: R¹—Y wherein R¹ is a member selected from the group consistingof optionally substituted (C₈-C₁₈)bicycloalkyl, optionally substituted(C₈-C₁₈)tricycloalkyl, optionally substituted (C₈-C₁₈)heterobicycloalkyland optionally substituted (C₈-C₁₈)heterotricycloalkyl; and Y is amember selected from the group consisting of a carboxylic acid, acarboxylate ester, a carboxylic acid chloride and other activated formsof carboxylic acids.
 7. A method in accordance with claim 1, whereinsaid solid support is selected from the group consisting of4-(bromomethyl)phenoxymethyl polystyrene, Merrifield resin, Rink amideresin and Sieber resin.
 8. A method in accordance with claim 4, whereinsaid acylating agent has the formula: R¹—Y wherein R¹ is a memberselected from the group consisting of optionally substituted(C₈-C₁₈)bicycloalkyl, optionally substituted (C₈-C₁₈)tricycloalkyl,optionally substituted (C₈-C₁₈)heterobicycloalkyl and optionallysubstituted (C₈-C₁₈)heterotricycloalkyl; and Y is a member selected fromthe group consisting of a carboxylic acid, a carboxylate ester, acarboxylic acid chloride and other activated forms of carboxylic acids.9. A method in accordance with claim 2, wherein said LXR ligands havethe formula:

wherein R¹ is a member selected from the group consisting of optionallysubstituted (C₈-C₁₈)bicycloalkyl, optionally substituted(C₈-C₁₈)tricycloalkyl, optionally substituted (C₈-C₁₈)heterobicycloalkyland optionally substituted (C₈-C₁₈)heterotricycloalkyl; R² is a memberselected from the group consisting of optionally substituted(C₁-C₈)alkyl, optionally substituted aryl, optionally substitutedheteroaryl, optionally substituted arylalkyl and optionally substitutedheteroarylalkyl; and X is a member selected from the group consisting of—CO₂R¹¹, —CH₂OR¹¹, —C(O)R¹¹, —C(O)NR¹¹R¹² and —CH₂NR¹¹R¹², wherein R¹¹and R¹² are each members independently selected from the groupconsisting of hydrogen and optionally substituted (C₁-C₈)alkyl.
 10. Amethod in accordance with claim 9, wherein R¹ is a member selected fromthe group consisting of optionally substituted optionally substitutedtricyclo[3.3.1.1^(3,7)]decanyl, optionally substitutedbicyclo[3.2.1]octanyl, optionally substituted bicyclo[5.2.0]nonanyl,bicyclo[4.3.2]undecanyl, optionally substitutedtricyclo[2.2.1.0¹]heptanyl, tricyclo[5.3.1.1¹]dodecanyl, optionallysubstituted tricyclo[5.4.0.0^(2,9)]undecanyl, optionally substitutedtricyclo[5.3.2.0^(4,9)]dodecanyl, optionally substitutedtricyclo[4.4.1.1^(1,5)]dodecanyl and optionally substitutedtricyclo[5.5.1.0^(3,11)]tridecanyl group.
 11. A method in accordancewith claim 9, wherein R¹ is a substituted or unsubstituted adamantylgroup.
 12. A method in accordance with claim 1, wherein said solidsupport is selected from the group consisting of a4-(bromomethyl)phenoxymethyl polystyrene and Merrifield resin; saidaniline derivative has the formula:

wherein PG is a protecting group, and said method further comprises astep between steps (a) and (b) of removing said protecting group; saidaldehyde or ketone of step (b) is selected from the group consisting ofa optionally substituted (C₁-C₅)alkyl aldehyde or ketone; and saidacylating agent of step (c) has the formula: R¹—Y wherein R¹ is a memberselected from the group consisting of optionallysubstituted(C₈-C₁₈)bicycloalkyl, optionallysubstituted(C₈-C₁₈)tricycloalkyl, optionallysubstituted(C₈-C₁₈)heterobicycloalkyl and optionallysubstituted(C₈-C₁₈)heterotricycloalkyl; and Y is a member selected fromthe group consisting of a carboxylic acid, a carboxylate ester, acarboxylic acid chloride and other activated forms of carboxylic acids.13. A method for preparing LXR ligands on a solid support, said methodcomprising: (a) attaching a substituted aniline derivative to said solidsupport to provide a support-bound substituted aniline derivative; and(b) contacting said support-bound substituted aniline derivative with anacylating agent to provide an LXR ligand on a solid support.
 14. Amethod in accordance with claim 13, further comprising: (c) removingsaid LXR ligand from said solid support.
 15. A method in accordance withclaim 13, wherein said substituted aniline derivative has the formula:

wherein PG is a protecting group; R² is a member selected from the groupconsisting of optionally substituted(C₁-C₈)alkyl, optionally substitutedaryl and optionally substituted heteroaryl; and said method furthercomprises a step between steps (a) and (b) of removing said protectinggroup.
 16. A method in accordance with claim 13, wherein said acylatingagent has the formula: R¹—Y wherein R¹ is a member selected from thegroup consisting of optionally substituted(C₈-C₁₈)bicycloalkyl,optionally substituted(C₈-C₁₈)tricycloalkyl, optionallysubstituted(C₈-C₁₈)heterobicycloalkyl and optionallysubstituted(C₈-C₁₈)heterotricycloalkyl; and Y is a member selected fromthe group consisting of carboxylic acid, carboxylate ester, carboxylicacid chloride and activated forms of carboxylic acids.
 17. A method inaccordance with claim 13, wherein said solid support is selected fromthe group consisting of a 4-(bromomethyl)phenoxymethyl polystyrene,Merrifield resin, Rink amide resin and Sieber resin.
 18. A method inaccordance with claim 15, wherein said acylating agent has the formula:R¹—Y wherein R¹ is a member selected from the group consisting ofoptionally substituted (C₈-C₁₈)bicycloalkyl, optionally substituted(C₈-C₁₈)tricycloalkyl, optionally substituted (C₈-C₁₈)heterobicycloalkyland optionally substituted (C₈-C₁₈)heterotricycloalkyl; and Y is amember selected from the group consisting of a carboxylic acid, acarboxylate ester, a carboxylic acid chloride and other activated formsof carboxylic acids.
 19. A method in accordance with claim 14, whereinsaid LXR ligands have the formula:

wherein R¹ is a member selected from the group consisting of optionallysubstituted(C₈-C₁₈)bicycloalkyl, optionally substituted(C₈-C₁₈)tricycloalkyl, optionally substituted (C₈-C₁₈)heterobicycloalkyland optionally substituted (C₈-C₁₈)heterotricycloalkyl; R² is a memberselected from the group consisting of optionally substituted(C₁-C₈)alkyl, optionally substituted aryl and optionally substitutedheteroaryl; and X is a member selected from the group consisting of—CO₂R¹¹, —CH₂OR¹¹, —C(O)R¹¹, —C(O)NR¹¹R¹² and —CH₂NR¹¹R¹², wherein R¹¹and R¹² are each members independently selected from the groupconsisting of hydrogen and optionally substituted (C₁-C₈)alkyl.
 20. Amethod in accordance with claim 13, wherein said substituted anilinederivative has the formula:

wherein PG is a protecting group; R² is a member selected from the groupconsisting of optionally substituted (C₁-C₈)alkyl, optionallysubstituted aryl and optionally substituted heteroaryl; and said methodfurther comprises a step between step (a) and (b) of removing saidprotecting group; and said acylating agent has the formula: R¹—Y whereinR¹ is a member selected from the group consisting of optionallysubstituted (C₈-C₁₈)bicycloalkyl, optionally substituted(C₈-C₁₈)tricycloalkyl, optionally substituted (C₈-C₁₈)heterobicycloalkyland optionally substituted (C₈-C₁₈)heterotricycloalkyl; and Y is amember selected from the group consisting of carboxylic acid,carboxylate ester, carboxylic acid chloride and activated forms ofcarboxylic acids.
 21. A combinatorial library comprising compounds ofthe formula

wherein R¹ is a member selected from the group consisting of optionallysubstituted(C₈-C₁₈)bicycloalkyl, optionally substituted(C₈-C₁₈)tricycloalkyl, optionally substituted (C₈-C₁₈)heterobicycloalkyland optionally substituted (C₈-C₁₈)heterotricycloalkyl; R² is a memberselected from the group consisting of optionally substituted(C₁-C₈)alkyl, optionally substituted aryl and optionally substitutedheteroaryl; and X is a member selected from the group consisting of—CO₂R¹¹, —CH₂OR¹¹, —C(O)R¹¹, —C(O)NR¹¹R¹² and —CH₂NR¹¹R¹², wherein R¹¹and R¹² are each members independently selected from the groupconsisting of a solid support, hydrogen and optionally substituted(C₁-C₈)alkyl.
 22. A method for synthesizing a combinatorial librarycomprising compounds of the formula:

wherein R¹ is a member selected from the group consisting of optionallysubstituted(C₈-C₁₈)bicycloalkyl, optionally substituted(C₈-C₁₈)tricycloalkyl, optionally substituted (C₈-C₁₈)heterobicycloalkyland optionally substituted (C₈-C₁₈)heterotricycloalkyl; R²is a memberselected from the group consisting of optionally substituted(C₁-C₈)alkyl, optionally substituted aryl and optionally substitutedheteroaryl; and X is a member selected from the group consisting of—CO₂R¹¹, —CH₂OR¹¹, —C(O)R¹¹, —C(O)NR¹¹R¹² and —CH₂NR¹¹R¹², wherein R¹¹and R¹² are each members independently selected from the groupconsisting of hydrogen and optionally substituted (C₁-C₈)alkyl; saidmethod comprising: (a) attaching an aniline derivative to a solidsupport to provide a support-bound aniline derivative; (b) contactingsaid support-bound aniline derivative with an aldehyde or ketone underreductively aminating conditions to provide a support-bound substitutedaniline derivative; and (c) contacting said support-bound substitutedaniline derivative with an acylating agent to provide an LXR ligand onsaid solid support.